1. Field of the Invention
Citrus bacterial canker disease (CBCD) is a serious disease of citrus, and the causal pathogen, Xanthomonas campestris pv. citri (Hasse) Dye (X.c. citri) is the subject of international quarantine. Although eradicated from the United States at great cost in the first third of this century, CBCD has reoccurred in several locations in Florida in recent years. A new eradication campaign has ensued, resulting in renewed interest in the disease and in methods for detecting the pathogen.
Several pathotypes within X.c. citri have long been recognized based on host range, geographical origin, bacteriophage sensitivities, plasmid content and serology. It is currently believed that at least three types of CBCD, types A, B and C, occur world-wide and are induced by variants of the same causal agent. These variants are primarily distinguished by their geographical origin and their host range. Pathotype A has both the widest host range and a global distribution. In addition, some pathogenic strains of X. campestris may also be associated with citrus bacteriosis in Mexico and have been described as the D type of the disease. Pathotypes B, C and D have so far been restricted to lemon (Citrus limon) and lime (Citrus aurantifolia) in South America and Mexico. The existence of the pathotypes has been confirmed at the DNA level by genomic fingerprinting (Hartung and Civerolo, 1987) and by restriction fragment length polymorphism (RFLP) analyses (Gabriel et al., 1988, and Hartung and Civerolo, 1989).
Another group of strains of X. campestris, which is genomically heterogeneous and different from X.c. citri, is associated with citrus bacterial spot disease (CBSD) in Florida. CBSD causes minor foliar damage primarily in citrus nurseries and is no longer considered a threat to the citrus industry. Strains of X. campestris that cause CBSD can be distinguished from strains of X.c. citri by the methods mentioned above as well as by symptomology (Schoulteis et al., 1987) and DNA/DNA hybridization (Egel et al., 1991).
Because of the magnitude of the perceived threat by X.c. citri to the citrus industry resulting in plant quarantines in both the United States and abroad, the development of diagnostic methodologies has been a high priority. Due to the occurrence of strains of X. campestris that cause CBSD on Citrus spp. and closely related plants, a high degree of specificity is now required.
This invention relates to diagnostic probes which are capable of detecting all four forms of CBCD and of distinguishing between the pathogens responsible for this disease and those causing CBSD. In addition, oligonucleotide primers useful for polymerase chain reaction (PCR)-based detection methods are also described. They may effectively be used by plant pathologists, microbiologists or regulatory agencies charged with the responsibility of detecting X.c. citri in interstate or international commerce.